embryonic development, hatching time and newborn juveniles
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Embryonic development, hatching time and newborn juveniles ofOctopus tehuelchus under two culture temperatures
Ramiro Braga, Silvina Van der Molen, Julián Pontones, NicolásOrtiz
PII: S0044-8486(20)31227-8
DOI: https://doi.org/10.1016/j.aquaculture.2020.735778
Reference: AQUA 735778
To appear in: Aquaculture
Received date: 19 April 2020
Revised date: 22 June 2020
Accepted date: 27 July 2020
Please cite this article as: R. Braga, S. Van der Molen, J. Pontones, et al., Embryonicdevelopment, hatching time and newborn juveniles of Octopus tehuelchus under twoculture temperatures, Aquaculture (2020), https://doi.org/10.1016/j.aquaculture.2020.735778
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© 2020 Published by Elsevier.
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Embryonic development, hatching time and newborn juveniles of Octopus
tehuelchus under two culture temperatures
Ramiro Bragaa,b
, Silvina Van der Molena, Julián Pontones
a,b, Nicolás Ortiz
a
aLaboratory of Cephalopods, Instituto de Biología de Organismos Marinos, IBIOMAR-
CONICET. Boulevard Brown 2915, (9120), Puerto Madryn, Chubut, Argentina.
bUniversidad Nacional de la Patagonia San Juan Bosco. Ciudad Universitaria, 25 de
Mayo km 4- (9000), Comodoro Rivadavia, Chubut, Argentina.
Corresponding author:
Nicolás Ortiz
Phone: +54 280 4883184
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Abstract
The development of cephalopods early life stages is strongly influenced by
environmental variables, especially temperature. Octopus tehuelchus (d’Orbigny, 1834)
is an Atlantic Patagonian fishery resource currently being studied as a new species for
cultivation; however it is not known how temperature modulates its early life stages. In
this work, egg masses were artificially incubated at 13 and 16°C under controlled
aquarium conditions. In each thermal treatment, the stages of embryonic development,
embryo morphometry and survival throughout embryogenesis, as well as
embryogenesis duration were recorded. After hatching, the morphological description of
the juveniles was achieved and survival time in starvation was calculated for both
temperatures. At 16°C the mean embryonic duration was 85 days shorter than at 13°C.
For both thermal treatments, the highest mortalities occurred up to the beginning of
organogenesis, and no significant differences in hatching success were observed. The
temperature also showed the potential to increase or decrease the juvenile performance
at the early post hatching period. This resulted in a significant reduction in size and
weight of new born juveniles at 13°C but also in an average increment of 7 days in their
survival in starvation when compared to octopus reared at 16°C. The chromatophore
pattern was similar for both thermal treatments and was characteristic of juveniles of
this species. The observed differences seem to show adaptive mechanisms that optimize
embryos and juveniles viability under the different environmental temperatures that can
be found in the northern Atlantic Patagonian coast. From a practical point of view, our
findings are important to define the biological parameters and associated procedures for
the cultivation of the early life stages of O. tehuelchus.
Keywords: cephalopod, incubation temperature, octopus culture, hatchlings
performance, octopus early life stages.
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1. Introduction
The knowledge of the biological aspects of cephalopod’s early life stages and a
precise understanding of the species responses to environmental factors is required to
achieve significant survival values under culture (Boletzky and Villanueva, 2014;
Oosthuizen et al., 2002; Sakurai et al., 1996; Uriarte et al., 2018; Vidal et al., 2002a).
From an ecological perspective, this information is relevant to understand the influence
of environmental factors on the population structure and dynamics of octopuses in wild
populations (Boletzky, 2003, 1989; Juárez et al., 2015; Villanueva et al., 2016).
Octopodidae species may exhibit a holobenthic or merobenthic mode of life, which
is determinant both for culture experiences and for a better understanding of species
distribution. In holobenthic species the full life cycle is associated with benthos (Iglesias
et al., 2014; Vidal et al., 2014; Villanueva et al., 2016): the hatched juveniles are large
and benthic, which favors their feeding and survival under controlled culture conditions
(Rosas et al., 2007; Solorzano et al., 2009; Vidal et al., 2014). In contrast, in
merobenthic octopuses, hatchlings are planktonic before they reach the benthic mode of
life (i.e. paralarvae) and the lack of appropriate food under culture conditions for the
planktonic stage gives rise to a high mortality rate that has so far hampered their scaling
production (Espinoza et al., 2017; Farías et al., 2016; Uriarte and Farías, 2014; Valverde
et al., 2019; Zúñiga et al., 2013).
Whether they are holobenthic or merobenthic species, temperature directly
influences the cephalopods' life cycle (Forsythe et al., 2001; Iglesias et al., 2014;
Schwarz et al., 2018; Villanueva et al., 2016). It is the main factor modulating the
duration of embryogenesis. Also, the thermal condition affects especially the embryo
development and growth, the yolk absorption, and the hatching rate (Boletzky, 1987;
Grigoriou and Richardson, 2008; Uriarte et al., 2016; Vidal et al., 2014). In several
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cephalopod species, the duration of embryogenesis can be shortened when the
incubation of the eggs is carried out at high temperatures within the species optimum
temperature range (Caamal-Monsreal et al., 2016; Uriarte et al., 2016, 2012).
Temperatures at the extremes of the thermal range generate morphological anomalies
and death of the embryos, low hatching success, and a deficiency in the use of inner
yolk in newborn hatchlings, causing low growth rates and short survival times (Rosa et
al., 2012; Vidal and Boletzky, 2014; Vidal et al., 2002b; Villanueva et al., 2003; Zheng
et al., 2014).
Of the few species of coastal octopods found in the Argentine Sea, the “Pulpito”
Octopus tehuelchus is the most studied, particularly its ecological, biological and
fisheries aspects (Fassiano et al., 2017; Iribarne et at., 1991; Ré, 1998; Storero and
Narvarte, 2013). This species endemic to South America is found in the Atlantic Ocean
between the south of Brazil (16°S) and the northern part of Argentine Patagonia (44°S),
and from the intertidal up to 100m deep (Narvarte et al., 2006; Ré, 1998; Ré and Ortiz,
2011; Storero et al., 2010). In North Patagonia coastal areas, in the southern limit of its
distribution, O. tehuelchus artisanal fishery has been carried out since the 1950s (Bocco
et al., 2019; Narvarte et al., 2006; Ré, 1998). Currently, this fishery is not only the
support of the fishermen community but also benefits the local gastronomy by
positively influencing the growth of the economy of the area (Narvarte et al., 2006; Ré,
1998). O. tehuelchus is a holobenthic species, with a small adult size (up to 150 g) and a
seasonal reproductive cycle (Ré, 1998; Storero et al., 2010). In North Patagonia, the
laying and brooding season runs from autumn to spring, frequently in shallow subtidal
areas (<20m) (Iribarne, 1990; Ré, 1998; Storero et al., 2010). The species is categorized
as simultaneous terminal spawner and its fecundity is low, with a maximum of 220 eggs
per laying. The egg clutches are generally attached to rocks or mollusks’ shells; they are
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conformed by large eggs (9-12 mm long x 3-5 mm width and a stalk 4-6 mm long),
arranged individually or in groups of two or three eggs, giving raise to large benthic
juveniles (Iribarne, 1990; Pujals, 1986; Ré, 1998; Storero et al., 2012).
Experiences carried out under controlled aquarium conditions indicate that O.
tehuelchus can adapt well to captivity (Berrueta et al. 2020; Iribarne et al., 1991; Klaich
et al., 2008, 2006). For example, Klaich et al. (2006) showed that at 15°C the rates of
instantaneous growth and daily feeding in adults and subadults were higher than at
10°C. Little has been documented on the early life stages of this species. Previous
attempts performed in aquariums by Ré (1989) suggested that the embryogenesis could
demand 120 days at 15°C, and that juveniles can survive in starvation up to 10 days.
However, there is a lack of studies regarding the embryonic development, juvenile
morphology and performance, and their relation to temperature.
The existing knowledge about the biological attributes of O. tehuelchus, its good
tolerance to captivity, the absence of a paralarvae stage, and its great acceptance in the
regional market, in addition to a global context with high demand for this type of
products (FAO, 2018; Vidal et al., 2014) generate great interest in the cultivation of this
species. The comprehension of how temperature modulates embryogenesis and newly
born juveniles is crucial to evaluate the biological culture feasibility and to define and
plan the conditions for the Octopus tehuelchus early life stages culture. In turn,
determining how temperature affects populations would help to understand the
distribution and abundance of this species in nature. Thereby, the aim of this work is to
determine and evaluate the effect of two thermal regimes on embryos development
time, and on the morphology and survival of embryos and newly hatched juveniles.
2. Materials and methods
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2.1. Capture and eggs conditioning
Octopus tehuelchus spawning females along with their egg clutches were collected
during the 2016 spawning period. Specimens and clutches were gathered at a depth of
10 m using traps in the northwest coast of Nuevo Gulf (Argentine) (42° 42'S 65° 36'
W), on the north Atlantic Patagonian coast. These traps were hoisted by diving and
checked on the vessel to verify the presence of eggs and spawning females. Traps with
spawning females and eggs were carried in aerated seawater to the CCT CONICET-
CENPAT (Centro Científico Tecnológico, Consejo Nacional de Investigaciones
Científicas y Técnicas - Centro Nacional Patagónico) Experimental Aquarium where the
specimens were acclimated for 24 hours.
2.2. Artificial incubation
Egg clutches were separated from females and the embryo development stages were
determined under a magnifying glass following Naef (1928). The 20 stages of Naef’s
embryonic scale encompass the blastulation (stages I-II) and gastrulation (stages III-VI)
processes, stage VII, which is considered a transitional phase, and the organogenesis
(stages VIII-XVI) and post-organogenesis (stages XVII-XX) processes. Five complete
egg clutches of 68, 44, 40, 64 and 40 eggs between stages I-II were selected to perform
the incubation experiments. The eggs in each clutch were randomly separated into two
groups with the same number of eggs and were artificially incubated in controlled
temperature chambers at two experimental temperatures: 13±1°C and 16±1°C (i.e. the
seawater temperatures found during the spawning and brooding period in the sampling
area) (Ortiz et al., 2011; Williams et al., 2018). For these experiments, five glass
aquariums with five liters of filtered (10, 5, and 1 µm), UV-sterilized, and aerated
seawater were used for each temperature. Water in the aquariums was partially replaced
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every two days and its quality was monitored daily. Dissolved oxygen was >5 mg/l, pH
range 7.5-8.4, total ammonia concentration lower than 1 mg L−1
and salinity of 33-35
ppt. The photoperiod was 12 h light: 12 h dark.
To allow a constant circulation of water over the eggs, proper visual inspection and
correct handling, each egg group was incubated in plastic mesh baskets. In the absence
of the females’ parental care, the eggs were cleaned by the same operator with a fine
brush (Ortiz and Ré, 2011; Ortiz et al., 2006) every three days, thus avoiding fouling
accumulation on the eggs’ chorion, which acts as a barrier between the embryo and the
external environment (Parra et al., 2000; Uriarte et al., 2012). During handling, the eggs
were held by the remaining cement of the egg stalk to avoid any kind of embryo
disturbance, either by breaking the stalk or the embryo chamber. Manual cleaning was
interrupted at stage XIX to prevent any premature hatching (Ortiz and Ré, 2011) that
may reduce survival chances (Boletzky and Villanueva, 2014; Vidal et al., 2014).
2.3. Embryos: morphological analysis, survival and development time
The eggs were monitored daily and potentially non-viable eggs (i.e. eggs with
abnormal development) were counted, assigned to an embryonic stage and separated in
an individual plastic mesh to verify the non-viability with time. Random samples of 5
eggs from each aquarium were observed every 7 days under a stereo microscope to
check the embryo development stage. From these data, the relative embryonic survival
(RES%) was calculated as RES% = nº viable eggs in stage i x 100 / nº viable eggs in
stage i-1, where i is the embryonic stage in which the non-viability of the egg was
registered. In addition, total embryo survival was evaluated, and the embryonic
development duration at each temperature treatment was recorded. Likewise, the
hatching period was defined as the difference in time between the first and the last
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hatching in the same laying (Vidal et al., 2014). In addition, every 10 days, eggs were
photographed under a stereomicroscope, and embryos’ morphological parameters (mm)
such as dorsal mantle length (DML), arm length (AL), eye diameter (ED) and outer yolk
sac volume (OYSV) were obtained as development indicators, with the ImageJ software
(Schneider et al., 2012). The OYSV (mm3) was calculated as OYSV=4πAB2/3, where A
is the half of the outer yolk sac length, and B is the half of the outer yolk sac width
(Uriarte et al., 2014; Vidal et al., 2002a).
2.4. Juveniles: morphological description and starvation survival time
For the morphological descriptions, a total of 33 one day post-hatching juveniles
from the 13°C treatment and 34 from the 16°C treatment were randomly sampled from
all aquariums. The juveniles were anesthetized using 96% ethanol, thus minimizing
stress (Butler-Struben et al., 2018). Ethanol was gradually added to the seawater,
starting at 0.5% v/v and increasing to 1 and 1.5% at 2 minute intervals until sedation.
Afterward, using the same methodology as with the embryos, the following
morphometric measurements (mm) were obtained: eye diameter (ED), mantle width
(MW), head width (HW), ventral mantle length (VML), total length (TL), dorsal mantle
length (DML), arm length (AL), and funnel length (FL). In addition, the total wet
weight (TW) (in grams), the chromatophore pattern, and the number of suckers were
recorded. The morphometric measurements and the description of juveniles were made
following Hochberg et al., (1992), Sweeney et al., (1992) and Young et al., (1989). For
observations with scanning electron microscopy (SEM), samples were fixed in 2.5%
glutaraldehyde in phosphate buffer for 3 hours and then dehydrated with the critical
point method for subsequent gold coating (Ortiz and Ré, 2011).
To calculate survival time in starvation, juveniles hatched in each thermal treatment
were separated by hatching date in three-liter plastic aquariums. A total of 62 juveniles
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distributed in 36 aquariums at 13°C, and 44 juveniles distributed in 21 aquariums at
16°C, were studied (density: 1 specimen/liter). Juveniles were kept under the same
temperature, photoperiod, and water quality conditions that they were incubated in. In
the rearing tanks, two pebbles per octopus were provided as refuge.
2.5. Data analysis
Throughout the embryonic development, each morphological parameter was
compared between and within temperature treatments. For this, a linear mixed effects
model was constructed (Pinheiro et al., 2016), where the morphometric parameters were
considered as the response variable, the temperature and embryogenesis time as fixed
explanatory variables and the aquarium as a random explanatory variable. A generalized
least squares model was used to evaluate the effect of the temperature on the
morphological parameters on the same day of embryogenesis. In turn, a Tukey test was
used to compare among embryogenesis days within each thermal treatment. To analyze
the effect of temperature and embryo development stages on RES%, a generalized
linear model of mixed effects (GLMM) (Zuur et al., 2009) with a binomial distribution
(link=logit) was performed, considering the aquarium as a random variable. To analyze
the effect of temperature on total embryonic survival, a generalized linear model (GLM)
with a quasi-binomial distribution (link=logit) was used. To evaluate the effect of
temperature on the survival time in starvation of juveniles, a GLM with a Gamma
distribution (link= inverse) was constructed. In this case, the aquarium was not used as a
random variable since it explained only 0.06 % of the total variance. For model
selection, the Akaike information criterion was used for each analysis. The effect of
temperature on morphological parameters of newborn juveniles was evaluated with a
one-way Anova. In addition, to assess the relationship between starvation survival time
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and hatching time, Pearson's correlation coefficient was calculated (Zar, 1996). The
aquariums were considered as replicates, except when comparing morphological
parameters of the newly born juveniles in which the specimens were considered as
replicates. Statistical analyzes were performed with the R statistical package, version
4.0.0, the level of significance used was p <0.05.
3. Results
3.1. Embryo development time and morphological description
Temperature strongly affects the embryogenesis of O. tehuelchus. Embryonic
development (mean ± SD) took 186 ± 8.68 days until the first hatching at 13°C and 101
±5.68 days at 16°C. The average (± SD) hatching period was 38 ± 12.5 days at 13°C
and 21 ± 12.58 days at 16°C. The time elapsed between the first and the last hatching
was 68 and 37 days at 13°C and 16°C respectively (Fig. 1). For the same incubation
time, embryos treated at 16°C presented a more advanced development stage than those
treated at 13°C. At 16°C, the progress of embryonic development seemed to be nearly
constant until hatching. However, at 13°C two phases could be distinguished: the first
one running from stages I-II up to the end of organogenesis at stages XV-XVI, and the
second, slower than the first, going from stages XV-XVI up to hatching (Fig. 1).
For both temperatures, during the first 30 days of embryonic development, most of
the eggs were in blastulation, with the formation of the germinal disc at the animal pole
(stage I-II) (Fig. 2A), or with the blastoderm cells migrating over the surface of the yolk
(gastrulation process) and covering the egg partially (Fig. 2B) or totally, a process that
occurs from stage III to stage VI. From day 45, at both temperatures most of the
embryos were at the vegetal pole (i.e. end of the first inversion). At this time the
perivitelline membrane encloses the embryo, the egg chamber becomes internally
separated from the stalk and the organ rudiments of the embryo (seen as an increase of
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distinct tissue concentration) begins to appear (i.e. beginning of organogenesis) (stage
VIII) (Fig. 2C, D). On day 50, all embryos under the higher temperature treatment were
between stages IX-X. In these stages, the retina pigment in the eyes is evident, the
mantle complex in the cephalic region is more developed, and more elongated arms
with the first recognizable suckers were observed (Fig. 2E). However, none of the
embryos subjected to 13°C exceeded stages VII-VIII (Fig. 1). On day 65, at 16°C some
of the embryos reached stage XIII-XIV, with the mantle and the arms more elongated,
the suckers more developed (Fig. 2F) and the optical complex in the form of a cup; at
13°C no embryo exceeded stages IX-X (Fig. 1). By day 75, most embryos subjected to
higher temperatures were between stages XV-XVI, where the first chromatophores
appear on the dorsal head, the eye becomes full pigmented and the systemic and
branchial heart is pulsating. Between days 90 and 95, most embryos at 16°C reached
stages XVII-XVIII. At stage XVIII, chromatophores begin to appear in mantle and arms
and the inner yolk sac become larger (Fig. 2G), while at 13°C most embryos are still at
stages IX-X. At 16°C most embryos reached stages XIX-XX between days 100 and
105, while at 13°C those stages were attained between days 180 and 200 (Fig. 1). At
stage XIX, the embryos return to the animal pole (end of the second inversion), and the
outer yolk sac is almost completely absorbed (Fig. 2H). After the complete absorption
of the external yolk, specimens would be ready to hatch. Not all embryos made the first
inversion, and complete their normal development directly at the animal pole side (Fig.
2F).
3.2. Embryos morphological analysis
The ED and AL were significantly affected by temperature and embryogenesis time,
while DML and OYSV were significantly affected by the interaction between both
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factors (Table 1). Significant differences were firstly observed on day 45 of embryonic
development when DML, ED, and AL were larger in the embryos subjected to higher
temperatures (Table 2, Fig. 3 A, B, C). For OYSV statistical differences were registered
at day 65, with lower volumes in embryos maintained at 16 than those at 13°C (Fig.
3D). These trends continued until the end of embryonic development (Table 2, Fig. 3).
Regarding comparisons within temperature treatments, embryos at 13°C showed a
significant increase in DML (F=51.91, p<0.0001), ED (F=18.01, p<0.0001) and AL
(F=73.91, p<0.0001) from day 75 on, while at 16°C this pattern was observed from day
45 onwards (DML: F=35.10, p<0.0001; ED: F=28.58, p<0.0001; AL: F=23.70,
p<0.0001). However, at 16ºC, the OYSV decreased significantly (F=35.32, p<0.0001)
from day 65 onwards, while at the lower temperature this was evident twenty days
before (F=38.10, p<0.0001).
3.3. Embryonic survival
Relative embryonic survival (RES%) was significantly affected only by the embryo
development stages (Chisq = 6.653, p = 0.0098). For both temperature treatments, the
highest mortality was recorded in the early stages of embryogenesis (Fig. 4). The lowest
RES% occurred between stages III and VI (88% and 86% for 13°C and 16°C,
respectively), and it increased between stages IX and XII until reaching its maximum
levels (100%). Towards the end of embryonic development (stages XIX-XX), the
RES% decreased slightly, reaching percentages of 96% at 16°C and 95% at 13°C (Fig.
4). Regarding total embryo survival, no significant differences were observed between
temperatures (Chisq=0.0257, p=0.8724) (Fig. 5), the average hatching success was
64%.
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3.4. Morphological features of juveniles
At hatching, the large O. tehuelchus juveniles completely adopt the benthic mode of
life, lying on the floor or hiding in shelters. No significant differences were found in the
DML, AL and FL. However, the ED, MW, HW, VML, TL and TW were significantly
higher in juveniles from the higher temperature treatment (Table 3).
In both thermal treatments, specimens presented their head, arms, interbrachial
membrane (arm webs), eyes, dorsal mantle, and posterior cap of the mantle densely
covered in small brown-reddish and black tegumental chromatophores. In the funnel,
the anterior margin of the ventral mantle and the perivisceral epithelium there was no
pigmentation. The mantle has a flask shape (Fig. 6). For each arm, a total of 50 suckers
along long stalks were counted. The two suckers closest to the mouth were arranged
linearly, whereas the rest were disposed in a zigzag row and decreased in size towards
the tip, which lacked terminal suckers. There were emerged fascicles of the Kölliker’s
organs spread over the skin of the arms, mantle and head (Fig. 7). These bristle-like
structures could also be seen as iridescent dots in living animals under incident light
(Fig. 6).
3.5. Juveniles’ survival time in starvation
Even when juveniles’ maximum survival time was similar between treatments (54
and 59 days at 13 and 16°C, respectively), the survival time in starvation was
significantly higher (Chisq = 5.6482, p = 0.0174) in juveniles subjected to the lower
temperature treatment compared to those under a higher temperature (24 and 17 days on
average at 13 and 16°C, respectively). For both temperatures, the Pearson correlation
coefficient between day of hatching and survival time in starvation was significant and
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negative (p<0.0001, r=-0.51 and p= 0.0109, r= -0.38 for 13 and 16 °C), this means that
the firsts to hatch survived longer (Fig. 8). No cannibalism was observed among
juveniles.
4. Discussion
Cephalopods’ embryo development time is characterized by the yolkiness of the eggs
and it is modulated by temperature (Boletzky, 2003). In general terms, small eggs
present a shorter embryonic development than larger ones, and species adapted to cold
water environments show longer embryogenesis duration than tropical ones (Boletzky,
1987, 1984; Clarke, 1982; Vidal et al., 2014). Furthermore, within the range of
temperature adaptation of cephalopod species, higher temperatures promote shorter
embryonic development time without affecting the hatching success (Boletzky, 2003;
Caamal-Monsreal et al., 2016; Uriarte et al., 2016). Beyond the thermal threshold,
embryos experience irreversible physiological damages (Pimentel et al., 2012; Repolho
et al., 2014; Sánchez-García et al., 2017). In Octopus tehuelchus, a temperate species
which lays large eggs, the results of the present study showed a long embryological
period, of around six months at 13°C, which can be reduced by half with a 3°C increase,
without showing significantly thermal-related effects on the relative nor the total
embryonic survival. Moreover, the average hatching success of O. tehuelchus (64%),
was similar to that found in artificial incubation experiences under optimal thermal
conditions for Enteroctopus megalocyathus (39.6%-65.8%), a species adapted to cold
water (Uriarte et al., 2016) and for O. maya (60-76%), a tropical species (Caamal-
Monsreal et al., 2016).
Under culture conditions, the period that goes from hatching up to stage VIII, when
organogenesis begins, seems to be critical for the survival of O. tehuelchus embryos,
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regardless culture temperature. During that period the blastulation and gastrulation
processes take place, and according to Boletzky (2003), the growth of the blastula is a
phase susceptible to malformations. Particularly, nonviable eggs at these stages
exhibited loss of turgidity, swelling and abnormal yellow coloring, as it was observed in
E. megalocyathus (Uriarte et al., 2011) and Loligo vulgaris reynaudii (Oosthuizen et al.,
2002). O. tehuelchus nonviable eggs also showed alterations such as a collapsed or
deformed (i.e. granulated or partially obliterated) yolk mass, which result in abnormal
morphogenesis and unviable embryos. In addition, during these development stages
there is no separation between the egg chamber and the egg stalk; this connection is
interrupted when the previtelline membrane is fully shaped at stage VIII. The
connection between the egg chamber and the stalk has been only described for O.
bimaculoides (Monsalvo-Spencer et al., 2013), a species that also produces large eggs
and benthic hatchlings. This particular embryological feature should be considered in
order to avoid O. tehuelchus embryonic mortality due to stalk break by wrong egg
handling before the beginning of organogenesis.
Temperature has a conditioning effect on the embryonic metabolic processes, such
as the rate and efficiency of yolk absorption, and growth of the embryos (Boletzky,
1987; Caamal-Monsreal et al., 2016; Uriarte et al., 2012; Vidal et al., 2002b; Villanueva
et al., 2007). In this sense, O. tehuelchus embryonic development showed different
patterns between incubation temperatures. While at 16°C embryo development rate
seems to be nearly constant throughout embryogenesis, at 13°C it slows down from
stages XV-XVI until hatching (i.e. post-organogenesis). Two similar phases were
observed in the embryo development pattern of L. vulgaris reynaudii (Oosthuizen et al.,
2002). From day 45 onwards, O. tehuelchus embryos at higher temperatures were larger
than those maintained at lower temperatures. On the other hand, at both temperatures, as
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a concomitantly effect of organogenesis processes, an increase in yolk consumption
became relevant at stages IX-X, indicating that embryos require greater amounts of yolk
to satisfy the high energy demands for organ formation and growth. These observations
agree with those for O. maya in which, regardless the incubation temperature, the
amount of outer yolk remained constant up to embryonic stage X, but the yolk
consumption rate increased afterwards (Camaal-Monsreal et al., 2016).
During the post-organogenesis period there are steep morphophysiological changes
during which embryos grow and go through the pre-hatching stages, acquiring the
competence to hatch (Boletzky, 2003; Naef, 1928; Olivares et al., 2019). Throughout
this period, the remaining outer yolk sac is transferred to the inner one and transfer rate
is influenced by temperature at which the embryo develops (Boletzky, 2003, 1994;
Bouchaud and Daguzan, 1990). The inner yolk sac acts as an energetic reserve during
the first days after hatching (Boletzky et al., 2002; Caamal-Monsreal et al., 2016; Naef,
1928; Sanchez-García et al., 2017). Since the rate of yolk absorption in O. tehuelchus
embryos subject to low temperatures slows down during the post-organogenesis stages,
almost duplicating the total development time, the speed of this process would
determine the individual hatching time and, consequently, the hatching period.
Moreover, the inverse relationship observed between hatching time and survival time
could be indicating an energetic tradeoff between the pre-hatching stages and hatchlings
performance. With a longer pre-hatching phase, the inner yolk would be consumed
inside the egg restricting the available energy during the first days of free life. In other
words, the processes occurring during the pre-hatching period would have a pivotal role
on the individual hatching time and the newborn juveniles of O. tehuelchus’
performance.
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The plasticity in the hatching time of cephalopods could represent an advantage for
their survival under heterogeneous environmental conditions (Boletzky, 2003).
According to Boletzky (1994, 1987), hatching could be delayed if embryonic
developmental temperature is close to the lower temperature limit to which the species
is adapted. A longer pre-hatching period might allow juveniles to hatch when food is
available, and thus it might have a regulatory function in places where food availability
shows seasonal fluctuations (Boletzky, 1994; Clarke, 1982). This would be the case in
temperate environments of the northern Atlantic Patagonia, at the southern end of the O.
tehuelchus distribution. In this area, water temperature ranges from 11 to 19°C and
primary production is strongly seasonal with two peaks, one in autumn and one in
spring (Rivas et al., 2006; Williams et al., 2018). Furthermore, the progressive and
discontinuous recruitment of juveniles observed between spring and early summer in
North Patagonia populations (Iribarne, 1991), could be explained by their temperature-
related hatching period that can spread from weeks to months, coupled with a laying
period that extends from early autumn to early winter (Iribarne, 1991; Ré, 1998; Storero
et al., 2010). On the contrary, a shorter embryonic development and hatching period due
to an increase in temperature would allow the octopuses to hatch earlier, grow faster and
survive to reproductive age, increasing population fitness (Nande et al., 2018; Uriarte et
al., 2012). This would be the case in the inner shelf of the subtropical waters of southern
Brazil, where water temperature ranges between 12 and 24°C and productivity is
relatively high (Ciotti et al., 1995), allowing the year-round availability of food for
hatchlings (Alves and Haimovici, 2011; Ciotti et al., 1995).
In this work, temperature showed the potential to increase or decrease the
performance of juveniles at the early post hatching period. Studies on Sepia officinalis,
L. opalescens and O. vulgaris recorded that hatchlings obtained at low temperatures
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were larger and heavier than those incubated at warmer temperatures (Bouchaud, 1991;
Repolho et al., 2014; Vidal et al., 2002a). In turn, Caamal-Monsreal et al. (2016)
established that in O. maya the best hatchling performance occurred in embryos
incubated at intermediate temperatures (22-26°C) and not at thermal extremes (18-
30°C). In O. tehuelchus, a higher incubation and juveniles’ maintenance temperature
seems to optimize the efficiency of yolk conversion, giving rise to larger juveniles, as is
reflected by cephalic measurements, TL, VML and in the wet weight. In contrast, at
lower temperatures the energy obtained through yolk absorption during embryonic
development would be used to guarantee basic hatchling metabolic processes and for
physiological maintenance, not for growth. Furthermore, at higher temperatures,
metabolic rates would be greater, so yolk absorption would be faster with the
subsequent reduction of survival time in starvation.
Since morphometric features can change with temperature and undergo changes
during the ontogeny of hatchlings, they would not be enough to perform the correct
identification of newly hatched juveniles from wild populations (Hochberg et al., 1992;
Kubodera and Okutani, 1981; Messenger, 2001; Young et al., 1989). In contrast,
chromatophore patterns are species-specific and remain unchanged throughout early
growth (Sweeney et al., 1992; Villanueva and Norman, 2008). Moreover, in O.
tehuelchus juveniles, chromatophore patterns seem showed to be independent of
temperature. In this sense, the presence of chromatophores densely covering the mantle,
heads and arms, and the absence of pigmentation in the funnel, might serve to
distinguish the newborn O. tehuelchus from other coastal sympatric species, such as
Enteroctopus megalocyathus and Robsonella fontaniana, already described in North
Patagonia (Ortiz and Ré, 2011; Ortiz et al., 2006).
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The differences observed between both experimental temperatures seem to show
adaptive mechanisms that optimize the viability of embryos and juveniles under the
different environmental temperatures that can be found in the northern Patagonian coast,
where O. tehuelchus has evolved. However, these compensatory mechanisms are
expected to trigger metabolic costs and behavioral changes on juveniles (Higgins et al.,
2012; Noyola et al., 2013a, 2013b) and hence, both consequences should be
investigated in order to set culture conditions for further rearing trials. From a practical
point of view, knowing the proper handling conditions of egg masses and the
temperature-related effects on embryogenesis and offspring survival would help
organize laboratory procedures and plan the availability of newborn juveniles of this
potential aquaculture resource at different times of the year.
Acknowledgements
This work was supported by the Agencia Nacional de Investigaciones Científicas y
Técnicas (PICT 210-0670). We want to thank Dr. Nicolás Battini for collaborating with
the juveniles’ photographs and for providing statistical advice, Dr. Augusto Crespi-
Abril for providing initial statistical advice, and Dr. Nora Glembocki for English
editing.
References
Alves, J., Haimovici, M., 2011. Reproductive biology of Octopus tehuelchus d’Orbigny,
1834 (Cephalopoda: Octopodidae) in southern Brazil. Nautilus (Philadelphia). 125,
150–158.
Journal Pre-proof
Jour
nal P
re-p
roof
20
Berrueta, M., Desiderio, J.A. Agliano, F., López, A.V, AristizabalAbud, E., Ortiz, N.,
2020. Mating behavior of Patagonian octopus (Octopus tehuelchus) under laboratory
conditions. Marine and Fishery Science. 33(1), 115-120.
Bocco, G., Cinti, A., Vezub, J., Sánchez-Carnero, N., Chávez, M., 2019. Lugar y
sentido de lugar en un camino de la costa atlántica patagónica, 1950-1970. Región Y
Soc. 31, e1127. doi:10.22198/rys2019/31/1127
Boletzky, S.V., 1984. Of The Octopus Scaeurgus unicirrhus (Mollusca,
Cephalopoda). Vie Milieu. 34, 87-93.
Boletzky, S.V., 1987. Embryonic phase, in: Boyle PR (Ed.), Cephalopod life cycles, vol
2. Acad Press., London, pp. 5-31.
Boletzky, S.V., 1989. Recent studies on spawning, embryonic development, and
hatching in the Cephalopoda. In Advances in Marine Biology, vol. 25. Acad Press.,
London, pp. 85-115.
Boletzky, S.V., 1994. Embryonic development of cephalopods at low temperatures.
Antarct. Sci. 6, 139–142. doi:10.1017/S0954102094000210
Boletzky, S.V., Fuentès, M., Offner, N., 2002. Developmental features of Octopus
macropus Risso, 1826 (Mollusca, Cephalopoda). Vie Milieu 52, 209–215.
Boletzky, S.V., 2003. Biology of early life stages in cephalopod molluscs. Adv. Mar.
Biol. 44, 143–203. doi:10.1016/S0065-2881(03)44003-0
Boletzky, S.V., Villanueva, R., 2014. Cephalopod Biology, in: Iglesias J., Fuentes, L.
Villanueva, R. (Eds.), Cephalopod Culture. Springer, Dordrecht, pp. 3-16.
Bouchaud, O., Daguzan, J., 1990. Etude expérimentale de l’influence de la température
sur le déroulement embryonnaire de la seiche Sepia officinalis L. (Céphalopode,
Sepioidae). Cah. Biol. Mar. 31, 131–145.
Journal Pre-proof
Jour
nal P
re-p
roof
21
Bouchaud, O., 1991. Energy consumption of the cuttlefish Sepia officinalis L.
(Mollusca: Cephalopoda) during embryonic development, preliminary
results. Bulletin of Marine Science, 49(1-2), 333-340.
Butler-Struben, H.M., Brophy, S.M., Johnson, N. A., Crook, R. J., 2018. In vivo
recording of neural and behavioral correlates of anesthesia induction, reversal, and
euthanasia in cephalopod molluscs. Front. Physiol. 9, 1–18.
doi:10.3389/fphys.2018.00109
Caamal-Monsreal, C., Uriarte, I., Farias, A., Díaz, F., Sánchez, A., Re, D., Rosas, C.,
2016. Effects of temperature on embryo development and metabolism of O. maya.
Aquaculture 451, 156–162. doi:10.1016/j.aquaculture.2015.09.011
Ciotti, Á.M., Odebrecht, C., Fillmann, G., Moller, O.O., 1995. Freshwater outflow and
Subtropical Convergence influence on phytoplankton biomass on the southern
Brazilian continental shelf. Cont. Shelf Res. 15, 1737–1756.
doi:10.1016/0278-4343(94)00091-Z
Clarke, A., 1982. Temperature and embryonic development in polar marine
invertebrates.Int. J. Invertebr. Repr, 5(2), 71-82.
doi:10.1080/01651269.1982.10553456
Espinoza, V., Viana, M.T., Rosas, C., Uriarte, I., Farías, A., 2017. Effect of starvation
on the performance of baby octopus (Robsonella fontaniana) paralarvae. Aquac. Res.
48, 5650–5658. doi:10.1111/are.13387
FAO. 2018. The State of World Fisheries and Aquaculture 2018 - Meeting the
sustainable development goals. Rome. Licence: CC BY-NC-SA 3.0 IGO.
Farías, A., Martínez-Montaño, E., Espinoza, V., Hernández, J., Viana, M.T., Uriarte, I.,
2016. Effect of zooplankton as diet for the early paralarvae of Patagonian red
Journal Pre-proof
Jour
nal P
re-p
roof
22
octopus, Enteroctopus megalocyathus, grown under controlled environment. Aquac.
Nutr. 22, 1328–1339. doi:10.1111/anu.12334
Fassiano, A.V., Ortiz, N., Ríos de Molina, M. del C., 2017. Reproductive status,
antioxidant defences and lipid peroxidation in Octopus tehuelchus (Cephalopoda:
Octopodidae) females. J. Nat. Hist. 51, 2645–2660.
doi:10.1080/00222933.2017.1329460
Forsythe, J.W., Walsh, L.S., Turk, P.E., Lee, P.G., 2001. Impact of temperature on
juvenile growth age at first egg-laying of the Pacific reef squid Sepioteuthis
lessoniana reared in captivity. Mar. Biol. 138, 103–112. doi:10.1007/s002270000450
Grigoriou, P., Richardson, C.A., 2008. The effect of ration size, temperature and body
weight on specific dynamic action of the common cuttlefish Sepia officinalis. Mar.
Biol. 154, 1085–1095. doi:10.1007/s00227-008-1002-3
Higgins, F.A., Bates, A.E., Lamare, M.D., 2012. Heat tolerance, behavioural
temperature selection and temperature-dependent respiration in larval Octopus
huttoni. J. Therm. Biol. 37, 83–88. doi:10.1016/j.jtherbio.2011.11.004
Hochberg, F.G., Nixon, M., Toll, R.B., 1992. Order OCTOPODA Leach, 1818.
Smithson. Contrib. to Zool. 513, 213-280.
Iglesias, J., Villanueva, R., Fuentes, L., 2014. Cephalopod culture. Springer, Dordrecht.
doi:10.1007/978-94-017-8648-5
Iribarne, O.O., 1990. Use of shelter by the small Patagonian octopus Octopus
tehuelchus : availability , selection and effects on fecundity. Mar. Ecol. Prog. Ser. 66,
251–258.
Iribarne, O.O., 1991. Life history and distribution of the small south-western Atlantic
octopus, Octopus tehuelchus. J. Zool. 223, 549–565.
doi:10.1111/j.1469-7998.1991.tb04387.x
Journal Pre-proof
Jour
nal P
re-p
roof
23
Iribarne, O.O., Fernandez, M.E., Zucchini, H., 1991. Prey selection by the small
Patagonian octopus Octopus tehuelchus d’Orbigny. J. Exp. Mar. Bio. Ecol. 148(2),
271–281. doi:10.1016/0165-7836(91)90020-G
Juárez, O.E., Galindo-Sánchez, C.E., Díaz, F., Re, D., Sánchez-García, A.M., Camaal-
Monsreal, C., Rosas, C., 2015. Is temperature conditioning Octopus maya fitness? J.
Exp. Mar. Bio. Ecol. 467, 71–76. doi:10.1016/j.jembe.2015.02.020
Klaich, M.J., Re, M.E., Pedraza, S.N., 2006. Effect of temperature, sexual maturity and
sex on growth, food intake and gross growth efficiency in the “Pulpito” Octopus
tehuelchus (D’orbigny, 1834). J. Shellfish Res. 25, 979–986.
doi:10.2983/0730-8000(2006)25[979:EOTSMA]2.0.CO;2
Klaich, M.J., Ré, M.E., Pedraza, S.N., 2008. Gross growth efficiency as a function of
food intake level in the “Pulpito” Octopus tehuelchus: A multimodel inference
application. Aquaculture 284, 272–276.
doi:10.1016/j.aquaculture.2008.07.054
Kubodera, T., Okutani, T., 1981. The systematics and identification of larval
Cephalopods from the northern north Pacific. Res. Inst. N. Pac. Fish. spe, 131–159.
McCullagh, P., Nelder, J.A., 1989. Generalized Linear Models. Monographs on
Statistics and Applied Probability Vol. 37. Taylor & Francis Group., New York.
Messenger, J.B., 2001. Cephalopod chromatophores: Neurobiology and natural history.
Biol. Rev. Camb. Philos. Soc. 76, 473–528.
doi:10.1017/S1464793101005772
Monsalvo-Spencer, P., Salinas-Zavala, C.A., Reynoso-Granados, T., 2013. Morfología
de la membrana coriónica de los huevos de Octopus bimaculoides y Octopus
hubbsorum (Cephalopoda: Octopodidae). Hidrobiologica 23, 124–129.
Naef, A., 1928. Die Cephalopoden. Embryologie. Die Fauna Flora Golf Neapel, 35(2),
Journal Pre-proof
Jour
nal P
re-p
roof
24
1-357.
Nande, A.M., Domingues, P., Rosas, C., 2018. Effects of Temperature on the
Embryonic Development of Octopus vulgaris 37, 1013–1019.
doi:10.2983/035.037.0512
Narvarte, M., González, R., Fernández, M., 2006. Comparison of Tehuelche octopus
(Octopus tehuelchus) abundance between an open-access fishing ground and a
marine protected area: Evidence from a direct development species. Fish. Res. 79,
112–119. doi:10.1016/j.fishres.2006.02.013
Noyola, J., Caamal-Monsreal, C., Díaz, F., Re, D., Sánchez, A., Rosas, C., 2013a.
Thermopreference, tolerance and metabolic rate of early stages juvenile Octopus
maya acclimated to different temperatures. J. Therm. Biol. 38, 14–19.
doi:10.1016/j.jtherbio.2012.09.001
Noyola, J., Mascaró, M., Caamal-Monsreal, C., Noreña-Barroso, E., Díaz, F., Re, D.,
Sánchez, A., Rosas, C., 2013b. Effect of temperature on energetic balance and fatty
acid composition of early juveniles of Octopus maya. J. Exp. Mar. Bio. Ecol. 445,
156–165. doi:10.1016/j.jembe.2013.04.008
Olivares, A., Rodríguez-Fuentes, G., Mascaró, M., Arteaga, A.S., Ortega, K., Monsreal,
C.C., Rosas, C., 2019. Maturation trade-offs in octopus females and their progeny:
energy, digestion and defence indicators. PeerJ, 7, e6618.
Oosthuizen, A., Roberts, M.J., Sauer, W.H.H., 2002. Temperature effects on the
embryonic development and hatching success of the squid Loligo vulgaris
reynaudii. Bull. Mar. Sci. 71, 619–632.
Ortiz, N., Ré, M.E., Márquez, F., 2006. First description of eggs, hatchlings and
hatchling behaviour of Enteroctopus megalocyathus (Cephalopoda: Octopodidae). J.
Plankton Res. 28, 881–890. doi:10.1093/plankt/fbl023
Journal Pre-proof
Jour
nal P
re-p
roof
25
Ortiz, N., Ré, M.E., 2011. The eggs and hatchlings of the octopus Robsonella
fontaniana (Cephalopoda : Octopodidae) 91, 705–713.
doi:10.1017/S0025315410001232
Ortiz, N., Ré, M.E., Márquez, F., Glembocki, N.G., 2011. The reproductive cycle of the
red octopus Enteroctopus megalocyathus in fishing areas of Northern Patagonian
coast. Fish. Res. 110, 217–223. doi:10.1016/j.fishres.2011.03.016
Parra, G., Villanueva, R., Yúfera, M., 2000. Respiration rates in late eggs and early
hatchlings of the common octopus, Octopus vulgaris.J. Mar. Biolog. Assoc. U.K.,
80(3), 557-558. doi:10.1017/S0025315400002319
Pimentel, M.S., Trübenbach, K., Faleiro, F., Boavida-Portugal, J., Repolho, T., Rosa,
R., 2012. Impact of ocean warming on the early ontogeny of cephalopods: A
metabolic approach. Mar. Biol. 159, 2051–2059. doi:10.1007/s00227-012-1991-9
Pinheiro, J.C., Bates, D.J., DebRoy, S., Sakar., D., R Core Team, 2016. Linear and
Nonlinear Mixed Effects Models. R package 3.1-128. URL: http://CRAN.R-
project.org/package=nlme.
Pujals, M. A., 1986. Contribución al conocimiento de la biología de Octopus tehuelchus
d'Orbigny (Mollusca: Cephalopoda). Anales de la Sociedad Científica Argentina,
Serie I, 214, 29-71.
Ré, M.E., 1989. Estudios ecológicos sobre el crecimiento y la alimentación de Octopus
tehuelchus d'Orbigny en Puerto Lobos, Golfo San Matías. Universidad Nacional de
La Plata
Ré, M.E., 1998. Pulpos octopódidos (Cephalopoda, Octopodidae), in: Boschi E.E. (Ed.),
El Mar Argentino y sus Recursos Pesqueros, Tomo 2. Instituto Nacional de
Investigación y Desarrollo Pesquero, Mar del Plata, pp69-98.
Journal Pre-proof
Jour
nal P
re-p
roof
26
Ré, M.E., Ortiz, N., 2011. Cefalópodos capturados en la Campaña “Concacen
noviembre 2009”, B/O Puerto Deseado. VIII Congreso Latinoamericano de
Malacología. Libro de resumenes. 249.
Repolho, T., Baptista, M., Pimentel, M.S., Dionísio, G., Trübenbach, K., Lopes,
V.M., Lopes, A.R., Calado, R., Diniz, M., Rosa, R., 2014. Developmental and
physiological challenges of octopus (Octopus vulgaris) early life stages under
ocean warming. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 184, 55–64.
doi:10.1007/s00360-013-0783-y
Rivas, A.L., Dogliotti, A.I., Gagliardini, D.A., 2006. Seasonal variability in satellite-
measured surface chlorophyll in the Patagonian Shelf. Cont. Shelf Res. 26, 703–720.
doi:10.1016/j.csr.2006.01.013
Rosa, R., Pimentel, M.S., Boavida-Portugal, J., Teixeira, T., Trübenbach, K., Diniz, M.,
2012. Ocean warming enhances malformations, premature hatching, metabolic
suppression and oxidative stress in the early life stages of a keystone squid. PLoS
One 7. doi:10.1371/journal.pone.0038282
Rosas, C., Cuzon, G., Pascual, C., Gaxiola, G., Chay, D., López, N., Maldonado, T.,
Domingues, P.M., 2007. Energy balance of Octopus maya fed crab or an artificial
diet. Mar. Biol. 152, 371–381. doi:10.1007/s00227-007-0692-2
Sakurai, Y., Bower, J.R., Nakamura, Y., Yamamoto, S., Watanabe, K., 1996. Effect of
temperature on development and survival of Todarodes pacificus embryos and
paralarvae. Am. Malacol. Bull. 13, 89–95.
Sanchez-García, A., Rodríguez-Fuentes, G., Díaz, F., Galindo-Sánchez, C.E., Ortega,
K., Mascaró, M., López, E., Caamal-Monsreal, C., Juárez, O., Noreña-Barroso, E.,
Re, D., Rosas, C., 2017. Thermal sensitivity of O. maya embryos as a tool for
monitoring the effects of environmental warming in the Southern of Gulf of Mexico.
Journal Pre-proof
Jour
nal P
re-p
roof
27
Ecol. Indic. 72, 574–585. doi:10.1016/j.ecolind.2016.08.043
Schneider, C.A., Rasband, W.S., Eliceiri, K.W., 2012. NIH Image to ImageJ: 25 years
of image analysis. Nat. Methods 9, 671–675. doi:10.1038/nmeth.2089
Schwarz, R., Piatkowski, U., Hoving, H.J.T., 2018. Impact of environmental
temperature on the lifespan of octopods. Mar. Ecol. Prog. Ser. 605, 151–164.
doi:10.3354/meps12749
Solorzano, Y., Viana, M.T., López, L.M., Correa, J.G., True, C.C., Rosas, C., 2009.
Response of newly hatched Octopus bimaculoides fed enriched Artemia salina:
Growth performance, ontogeny of the digestive enzyme and tissue amino acid
content. Aquaculture 289, 84–90. doi:10.1016/j.aquaculture.2008.12.036
Storero, L.P., Narvarte, M.A.,González, R.A., 2012. Reproductive traits of the small
Patagonian octopus Octopustehuelchus. Helgol. Mar. Res. 66, 651–659.
doi:10.1007/s10152-012-0298-z
Storero, L.P., Ocampo-Reinaldo, M., González, R.A., Narvarte, M.A., 2010. Growth
and life span of the small octopus Octopus tehuelchus in San Matías Gulf
(Patagonia): Three decades of study. Mar. Biol. 157, 555–564.
doi:10.1007/s00227-009-1341-8
Storero, L.P., Narvarte, M.A., 2013. Coccidian infection may explain the differences in
the life history of octopus host populations. J. Invertebr. Pathol. 114, 222–225.
doi:10.1016/j.jip.2013.08.006
Sweeney, M.J., Roper, C.F., Mangold, K.M., Clark, M.R., Boletzky, S.V., 1992.
"Larval" and juvenile cephalopods: a manual for their identification. Smithsonian
Contribution to Zoology 513. SmithsonianInstitutionPress, Washington, DC:
Uriarte, I., Iglesias, J., Domingues, P., Rosas, C., Viana, M.T., Navarro, J.C., Seixas, P.,
Vidal, E., Ausburger, A., Pereda, S., Godoy, F., Paschke, K., Farías, A., Olivares, A.,
Journal Pre-proof
Jour
nal P
re-p
roof
28
Zuñiga, O., 2011. Current Status and Bottle Neck of Octopod Aquaculture: The Case
of American Species. J. World Aquac. Soc. 42, 735–752. doi:10.1111/j.1749-
7345.2011.00524.x
Uriarte, I., Espinoza, V., Herrera, M., Zúñiga, O., Olivares, A., Carbonell, P., Pino, S.,
Farías, A., Rosas, C., 2012. Effect of temperature on embryonic development of
Octopus mimus under controlled conditions. J. Exp. Mar. Bio. Ecol. 416–417, 168–
175. doi:10.1016/j.jembe.2012.03.003
Uriarte, I., Espinoza, V., Gutiérrez, R., Zúñiga, O., Olivares, A., Rosas, C., Pino, S.,
Farías, A., 2014. Key aspects of egg incubation in Patagonian red octopus
(Enteroctopus megalocyathus) for cultivation purposes. Aquaculture 424–425, 158–
166. doi:10.1016/j.aquaculture.2013.12.039
Uriarte, I., Farías, A., 2014. Enteroctopus megalocyathus, in: Iglesias J., Fuentes, L.
Villanueva, R. (Eds.), Cephalopod Culture. Springer., Dordrecht, pp. 365-382.
Uriarte, I., Martínez-Montaño, E., Espinoza, V., Rosas, C., Hernández, J., Farías, A.,
2016. Effect of temperature increase on the embryonic development of Patagonian
red octopus Enteroctopus megalocyathus in controlled culture. Aquac. Res. 47,
2582–2593. doi:10.1111/are.12707
Uriarte, I., Rosas, C., Espinoza, V., Hernández, J., Farías, A., 2018. Thermal tolerance
of paralarvae of Patagonian red octopus Enteroctopus megalocyathus. Aquac. Res.
49, 2119–2127. doi:10.1111/are.13666
Valverde, J.C., Granero, M.D., Giménez, F.A., García, B.G., 2019. Successful rearing
of common octopus (Octopus vulgaris) fed a formulated feed in an offshore cage
968–972. doi:10.1111/are.13955
Journal Pre-proof
Jour
nal P
re-p
roof
29
Vidal, E.A.G., DiMarco, F.P., Wormuth, J.H., Lee, P.G., 2002a. Influence of
temperature and food availability on survival, growth and yolk utilization in
hatchling squid. Bulletin of Marine Science, 71(2), 915-931.
Vidal, E.A.G., DiMarco, F.P., Wormuth, J.H., Lee, P.G., 2002b. Optimizing rearing
conditions of hatchling loliginid squid. Mar. Biol. 140, 117–127.
doi:10.1007/s002270100683
Vidal, E.A.G., Boletzky, S.V., 2014. Loligo vulgaris and Doryteuthis opalescens. In
cephalopod culture. Springer., Dordrecht. pp. 271-313.
Vidal, E.A.G., Villanueva, R., Andrade, J.P., Gleadall, I.G., Iglesias, J., Koueta, N.,
Rosas, C., Segawa, S., Grasse, B., Franco-Santos, R.M., Albertin, C.B., Caamal-
Monsreal, C., Chimal, M.E., Edsinger-Gonzales, E., Gallardo, P., Le Pabic, C.,
Pascual, C., Roumbedakis, K., Wood, J., 2014. Cephalopod culture: Current status of
main biological models and research priorities, Advances in Marine Biology.
doi:10.1016/B978-0-12-800287-2.00001-9
Villanueva, R., Arkhipkin, A., Jereb, P., Lefkaditou, E., Lipinski, M.R., Perales-Raya,
C., Riba, J., Rocha, F., 2003. Embryonic life of the loliginid squid Loligo vulgaris:
Comparison between statoliths of Atlantic and Mediterranean populations. Mar.
Ecol. Prog. Ser. 253, 197–208. doi:10.3354/meps253197
Villanueva, R., Moltschaniwskyj, N.A., Bozzano, A., 2007. Abiotic influences on
embryo growth: Statoliths as experimental tools in the squid early life history. Rev.
Fish Biol. Fish. 17, 101–110. doi:10.1007/s11160-006-9022-x
Villanueva, R., Norman, M.D., 2008. Biology of the planktonic stages of benthic
octopuses. Oceanogr. Mar. Biol. 46, 105–208.
doi:10.1201/9781420065756.ch4
Villanueva, R., Vidal, E.A.G., Fernando, A., 2016. Early Mode of Life and Hatchling
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30
Size in Cephalopod Molluscs: Influence on the Species Distributional Ranges. PLoS
ONE 11(11): e0165334. doi:10.1371/journal.pone.0165334
Williams, G.N., Solís, M.E., Esteves, J.L., 2018. Satellite-measured phytoplankton and
environmental factors in north Patagonian Gulfs. Plankt. Ecol. Southwest. Atl. From
Subtrop. to Subantarctic Realm 307–325.
doi:10.1007/978-3-319-77869-3_15
Young, R.E., Harman, R.F., Hochberg, F.G., 1989. Octopodid paralarvae from
Hawaiian waters. The Veliger, 32(2), 152-165.
Zar, J.H., 1996. Biostatisical analysis. Prentice-Hall, Upper Saddle River, New Jersey
Zheng, X.D., Qian, Y.S., Liu, C., Li, Q., 2014. Octopus minor, in: Iglesias J., Fuentes,
L. Villanueva, R. (Eds.), Cephalopod Culture. Springer, Dordrecht, pp. 415-426.
Zúñiga, O., Olivares, A., Rojo, M., Chimal, M.E., Díaz, F., Uriarte, I., Rosas, C., 2013.
Thermoregulatory behavior and oxygen consumption of Octopus mimus
paralarvae: The effect of age. J. Therm. Biol. 38, 86–91.
doi:10.1016/j.jtherbio.2012.11.003
Zuur, A., Ieno, E.N., Walker, N., Saveliev, A.A.,Smith, G.M., 2009. Mixed effects
models and extensions in ecology with R. Springer Sci. Bus.Media. doi:10.1007/978-
0-387-87458-6_1
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Figure captions
Fig.1. Octopus tehuelchus embryo development stages versus time at 13°C (gray
squares) and 16°C (black squares). Each square represents the mode of 25 observations,
vertical lines show the maximum and minimum embryonic stages observed. Horizontal
lines indicate the hatching period at 13°C (gray line) and 16°C (black line).
Fig.2. Octopus tehuelchus embryo development stages. A) Stages I-II, B) Stages V-VI:
blastoderm cells occupying nearly 30-50% of the egg, C-D) Stage VIII: end of the first
inversion and beginning of organogenesis, E) Stages IX-X, F) Stage XIII-XIV, G) Stage
XVIII, H) Stage XIX: end of the second inversion. (a) arms, (ap) animal pole, (apr) arm
primordium, (bd) blastoderm, (c) chorion, (chr) chromatophores, (dh) dorsal side of
head, (dm) dorsal side of mantle, (e) eye, (gd) germinal disk, (iys) inner yolk sac, (m)
mantle complex, (mp) mantle primordium, (op) optic vesicle, (oys) outer yolk sac, (pm)
perivitelline membrane, (su) suckers, (y) yolk, (vp) vegetal pole. Note that the embryo
in image F did not perform the 1st inversion.
Fig.3. Relationship between embryo structures and age of Octopus tehuelchus reared at
13°C (grey squares) and 16°C (black squares). a) Dorsal mantle length, b) eye diameter,
c) arm length, and d) outer yolk sac volume. The vertical bars indicate the standard
error.
Fig.4. Octopus tehuelchus mean relative embryo survival (RES%) as a function of
embryo development stages (grey bars: 13°C, black bars: 16°C).
Fig.5. Mean (± SE) total embryo survival as a function of temperature (grey bars: 13
°C, black bars: 16 °C).
Fig.6. Lateral view of a newly hatched juvenile of Octopus tehuelchus, showing the
dorsal and ventral mantle, head, arms and eyes densely covered with chromatophores,
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and the funnel and anterior margin of the ventral mantle without a chromatophore cover.
Note the Kölliker´s organs seen as iridescent dots in the ventral mantle.
Fig.7. Kölliker’s organ detail from scanning electron micrograph on the mantle of
Octopus tehuelchus juvenile.
Fig.8. Octopus tehuelchus juveniles’ survival time in starvation, depending on the
hatching time at 13 °C (white circles) and 16 °C (black triangles).
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Ramiro Braga: Conceptualization, Methodology, Investigation, Formal analysis, Writing -
Original Draft, Visualitation, Writing - Review & Editing.
Silvina Van der Molen: Conceptualization, Investigation, Resources, Writing - Review & Editing,
Project administration, Funding acquisition.
Julián Pontones: Investigation, Formal analysis.
Nicolás Ortiz: Conceptualization, Methodology, Validation, Investigation, Resources,
Supervision, Formal analysis, Writing - Review & Editing, Project administration, Funding
acquisition.
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Declaration of interests
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
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Table 1. Results of the Linear mixed-effects model. Effect of temperature,
embryogenesis time, their interaction, and the aquarium as a random effect on embryos
morphometric parameters of Octopus tehuelchus.
DML ED AL OYSV
Fixed effects F-value p-value F-value p-value F-value p-value F-value p-value
Temperature 11.710 0.0091 7.613 0.0247 3.909 0.0534 1.066 0.3319
Embryogenesis
time 504.606 <.0001 176.627 <.0001 413.480 <.0001 205.219 <.0001
Temp*Embryo.
time 8.853 0.0034 2.132 0.1463 0.700 0.4046 28.091 <.0001
Random
effects L.Ratio p-value L.Ratio p-value L.Ratio p-value L.Ratio p-value
Aquarium 47.266 <.0001 27.751 <.0001 35.393 <.0001 0.299 0.584
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Table 2. Results of the Generalized least squares model. Effect of temperature on the
embryos morphometric parameters of Octopus tehuelchus throughout the
embryogenesis time.
Embryogenesis DML ED AL OYSV
time (days) F-value p-value F-value p-value F-value p-value F-value p-value
25 1.179 0.2988 1.019 0.3325 2.306 0.1547 0.447 0.5163
45 7.755 0.0127 12.015 0.003 7.581 0.0136 0.088 0.7703
55 24.521 1e-04
12.667 0.0022 15.089 0.0015 0.001 0.9662
65 72.288 <.0001 18.084 1e-04
55.163 <.0001 11.641 0.0015
75 126.148 <.0001 61.941 <.0001 67.341 <.0001 133.962 <.0001
95 12.800 0.0117 25.937 0.0022 19.045 0.0073 12.222 0.0129
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Table 3. Average (mm or g, +/- SE) of the different morphological variables of
anesthetized Octopus tehuelchus at hatching, and comparison of the morphological
variables between temperatures.
Morphological measures Incubation temperatures Statistical parameters
16°C 13°C F-value p-value
Number of juveniles 34 33
Eye diameter (ED) 1.48 ± 0.31 1.23 ± 0.18 4.79 0.0413
Mantle width (MW) 4.62 ± 0.31 4.27 ± 0.35 6.08 0.0234
Head width (HW) 4.95 ± 0.28 4.26 ± 0.45 18.22 0.0004
Ventral mantle length (VML) 5.05 ± 0.96 3.93 ± 0.49 11.11 0.0035
Total length (TL) 17.35 ± 1.43 15.78 ± 1.80 4.92 0.0389
Total weight (TW) 0.18 ± 0.03 0.16 ± 0.03 4.58 0.0368
Arm length (AL) 8.46 ± 1.44 8.15 ± 1.41 0.25 0.6249
Funnel length (FL) 1.87 ± 0.45 1.73 ± 0.42 0.54 0.4720
Dorsal mantle length (DML) 6.15 ± 0.59 5.87 ± 0.64 1.12 0.3039
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Highlights
1) Embryonic survival increases when the gastrulation process comes to an end.
2) Temperature influences the embryonic development pattern and embryos
morphometrics.
3) Temperature determines the individual hatching time and the hatching period.
4) The first octopuses to hatch survive longer.
5) Temperature could increase or decrease Octopus tehuelchus hatchlings performance.
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